Tunicamycin,puromycin and brefeldin A influence the subcellular distribution of neuropeptides in hypothalamic magnocellular neurones of rat

Summary Magnocellular neurones in the supraoptic nuclei of normal Long Evans and homozygous Brattleboro rats were examined electron-microscopically after intracisternal injections of tunicamycin, puromycin, or brefeldin A. Moderate (50 g) or high (200 g) doses of tunicamycin caused the formation of electron-dense filamentous accretions in the endoplasmic reticulum (ER) cisterns of vasopressin neurones, but only the high dose of tunicamycin also caused accretions to form in the ER of some oxytocin neurones. Immunogold labelling of ultrathin sections from tunicamycin-treated rats revealed that, in about 5% of vasopressin neurones, the accretions could be immunogold-labelled for vasopressin and its associated neurophysin. However, in the majority of vasopressin neurones, the sections required trypsinisation before immunolabelling of the accretions could be detected. Small accretions in the ER of oxytocin neurones did not label for oxytocin or its neurophysin without prior trypsinisation, whereas larger accretions in other oxytocin cells could be labelled without prior trypsin treatment. Administration of puromycin resulted in the formation of small ER accretions in both vasopressin and oxytocin neurones. These accretions were immunolabelled with antisera, respectively, to vasopressin and oxytocin, but neurophysin-immunoreactivity was in most cases absent and was not revealed by treatment with trypsin, suggesting that neurophysin-immunoreactive epitopes were absent from truncated peptides forming the accretions. Brefeldin A caused dilatation of ER cisterns and disruption of the Golgi apparatus in both oxytocin and vasopressin neurones, but did not cause accretions to form in the ER.     

Structure and sequence of the human α- -iduronidase gene

In humans, a deficiency of the lysosomal hydrolase α- -iduronidase (IDUA; EC 3.2.1.76) results in the lysosomal storage of the glycosaminoglycans heparan sulfate and dermatan sulfate, thereby causing the lysosomal storage disorder mucopolysaccharidosis type I. The gene for IDUA is split into 14 exons spanning approximately 19 kb. We report the sequence of two noncontiguous segments of the IDUA gene, one 1.8-kb segment containing exons 1 and 2 and surrounding sequences and a second segment of 4.5 kb containing the last 12 exons. The potential promoter for IDUA has only GC box type consensus sequences consistent with a housekeeping promoter and is bounded by an Alu repeat sequence. The first two exons of IDUA are separated by an intron of 566 bp, then there is a large intron of approximately 13 kb, and the last 12 exons are clustered within 4.5 kb. No consensus polyadenylation signal was found in the 3′ untranslated region, although two variant polyadenylation signals are proposed.     

Stereoselective effect of morphine on antinociception and endogenous opioid peptide levels in plasma but not cerebrospinal fluid of dogs.

Morphine releases endogenous opioids into the circulation of dogs. To test the stereospecificity of this effect, as well as to determine whether morphine also releases endogenous opioids centrally, which might be involved in its antinociceptive action, the effects of (-)-morphine sulfate (10 mg/kg, sc) or (+)-morphine hydrobromide on antinociception in a dog tail-flick test, on semi-quantified morphine-induced signs of salivation, emesis, defecation and ataxia, and on the plasma and cerebrospinal fluid (CSF) levels of endogenous opioid peptides were studied. Plasma and CSF levels of immunoreactive beta-endorphin (i-BE), met-enkephalin (i-ME), leu-enkephalin (i-LE), and dynorphin (i-DY) were quantified by radioimmunoassay in octadecylsilyl-silica cartridge extracts. Immunoreactive morphine (i-M) levels were measured in unextracted samples. (-)-Morphine treatment significantly increased antinociception, morphine-induced signs, i-M levels in plasma and CSF, and i-BE, i-ME, and i-LE levels in plasma, but not CSF. Levels of i-DY remained constant in plasma and CSF. (+)-Morphine treatment did not alter any of these parameters, indicating that the effects of morphine on nociception, behavioral signs, and plasma endogenous opioids in dogs were stereoselective. It is concluded that morphine does not cause an increase in immunoreactive endogenous opioid peptides in the CSF at the time of its peak antinociceptive effect.     

Structure and function of tyrosine kinase receptors

Over the past ten years, several growth factor receptors have been shown to be ligand-regulated tyrosine kinases. Tyrosine kinase activity is essential for signal transmission, suggesting that phosphorylation cascades may play an important role. Considerable effort has gone into understanding the structure and function of tyrosine kinase receptors in order to define their mechanisms of signal transmission. However, the protein substrates of the receptor kinases have proven to be difficult to isolate and clone. This review focuses on the receptors for insulin, epidermal growth factor, and platelet-derived growth factor. They are all tyrosine kinases, but emerging evidence suggests that they utilize multiple separate signal transduction pathways. Work carried out during the next several years should yield considerable insight into the complexity of the components which interact with these tyrosine kinase receptors to regulate cellular growth and metabolism.     

Human N-acetylgalactosamine-4-sulphatase: protein maturation and isolation of genomic clones

N-Acetylgalactosamine-4-sulphatase (EC 3.1.6.1, G4S) is composed of a 57 kDa species in human liver that dissociates into 43 kDa and 8 kDa subunits under reducing conditions and, when deficient, causes the lysosomal storage disorder, mucopolysaccharidosis type VI. We isolated genomic clones containing the G4S first exon, including the leader peptide and the amino terminus of the 43 kDa polypeptide. Amino-terminal amino acid sequences of the 43 kDa and 8 kDa subunits indicated that the 8 kDa component is linked to the 43 kDa polypeptide by a single disulphide bond, does not contain the mannose-6-phosphate lysosomal targeting signal and is at the carboxyl terminus of G4S.     

Nucleotide sequence of the LYS2 gene of Saccharomyces cerevisiae: homology to Bacillus brevis tyrocidine synthetase 1

The Saccharomyces cerevisiae LYS2 gene, which encodes alpha-aminoadipate reductase, an essential enzyme in the yeast lysine biosynthetic pathway, has been sequenced. A large open reading frame (ORF) has been identified which can specify a 1392-amino acid protein with a deduced Mr of 155,344. A DNA database search using the translated LYS2 ORF as a probe has revealed significant aa sequence homology to the Bacillus brevis enzyme tyrocidine synthetase 1.     

Assay of inorganic sulfate in biologic fluids by nonsuppressed (single-column) ion chromatography

An assay using nonsuppressed (single-column) anion chromatography was developed to determine the concentration of inorganic sulfate in biologic fluids. A conventional HPLC system with an anion-exchange column and conductimetric detector interfaced with an automatic injector and integrator was used. The mobile phase for the chromatography of urine and serum samples is 4 mM potassium hydrogen phthalate, pH 4.5, and potassium iodide is used as the internal standard. For cerebrospinal fluid samples, the mobile phase is modified by addition of 10% of a 4 mM phthalic acid solution. Results of the HPLC assay were found to correlate well (r = 0.991 and 0.999) with those of two commonly used spectrophotometric methods for urine and serum inorganic sulfate determinations. However, the concentrations determined by ion chromatography were 2.5 to 10% lower, possibly due to less assay interference by other substances following chromatographic separation of sulfate. Anion chromatography using a single-column system is a convenient and relatively inexpensive method with sufficient sensitivity for the determination of inorganic sulfate concentrations in urine, serum, and cerebrospinal fluid.     

Single amino acid substitutions in the reactive site of antithrombin leading to thrombosis. Congenital substitution of arginine 393 to cysteine in antithrombin Northwick Park and to histidine in antithrombin Glasgow

Antithrombin Northwick Park and antithrombin Glasgow are functionally variant antithrombins with impaired abilities to interact with thrombin. Thrombosis is associated with their inheritance. Both of the purified, reduced, and S-carboxymethylated variant antithrombins were treated with cyanogen bromide and the major pools of each containing the amino acid sequence Gly339-Met423 were isolated. Following treatment of these pools with trypsin, fast atom bombardment mass spectrometry identified tryptic peptides (found also in normal antithrombin treated in the same way) that corresponded to amino acid sequences Gly339-Lys370 and Val400-Met423. The tryptic peptides, corresponding to amino acid sequences Ala371-Arg393 and Ser394-Arg399 were present in both variant preparations in greatly reduced amounts compared to a normal antithrombin preparation. However, two novel tryptic peptides of molecular mass (M + H)+ 2976 and 2952 were identified in the digests of antithrombin Northwick Park and Glasgow, respectively. Further analyses of these novel tryptic peptides were carried out by V8 protease treatment and sequential Edman degradation coupled with mass spectrometric analysis of the shortened peptides. This established that these peptides comprised the amino acid sequence Ala371-Arg399, but with single amino acid substitutions at the reactive site, Arg393 replaced by Cys (in antithrombin Northwick Park) and by His (in antithrombin Glasgow).     

Design of an indicator of intracellular free Na+ concentration using 19F-NMR

The development is described of an Na+ chelator with appropriate properties for an indicator of intracellular free Na+ concentration ([Na+]i). The new indicator, FCryp-1, is a tribenzo derivative of the parent (2:2:1) cryptand structure, incorporating the same F-substituted dibenzo 19F-NMR reporter group as the free [Ca2+] indicator, 5FBAPTA (Smith, G.A., Hesketh, T.R., Metcalfe, J.C., Feeney, J. and Morris, P.G. (1983) Proc. Natl. Acad. Sci., USA 80, 7178-7182). FCryp-1 has appropriate affinity for Na+ (KNa = 10(1.3) M-1) and selectivity over other intracellular cations (KK; KCa; K Mg less than 10(-1) M(-1)) for a [Na]i indicator. There is an 19F-NMR chemical shift of 2.00 ppm between free FCryp-1 and the Na-FCryp-1 complex which provides a direct read out of free [Na+]. FCryp-1 carries four carboxylate groups to confer aqueous solubility which can be esterified with acetoxymethyl groups to render the indicator membrane permeant. Experiments on pig lymphocytes loaded with FCryp-1 gave an indicated [Na+]i of 13.8 +/- 1.8 mM (n = 4). The FCryp-1 structure can also be readily modified to provide fluorescent [Na+]i indicators.